Analyte injection system

ABSTRACT

This invention provides methods and systems for injection of analytes into a separation channel for resolution and detection. Samples can be preconditioned and concentrated by isotachophoresis (ITP) before the injection is triggered by a detected voltage event. Separation of analytes from other sample constituents can be enhanced using skewing channel ITP.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/500,387, filed Sep. 5, 2003, and U.S. Provisional Application No.60/518,169, filed Nov. 7, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the field of analytical electrophoresissystems and methods. The invention can include high resolution andhighly sensitive Isotachophoresis (ITP) and capillary electrophoresis(CE) assays.

2. Description of the Related Art

Electrophoresis is generally the movement of charged molecules in anelectric field. Analytical methods based on electrophoresis have foundbroad utility, especially in the fields of protein and nucleic acidanalyses. Samples having charged analyte molecules of interest can beplaced in a selective media, such as size exclusion media, ion exchangemedia, or media having a pH gradient, where they can differentiallymigrate for high resolution from other sample molecules. The separatedmolecules can be detected for identification and quantitation.

Capillary and microfluidic scale electrophoretic separations areparticularly popular for analyses requiring low sample volumes or highthroughput. For example, chips of plastic or glass substrate can befabricated with microscale loading channels, separation channels anddetection channels. Samples can be transferred from microwell platesthrough a robotically manipulated sample collection tube to the loadingchannel. An electric potential can induce movement of sampleconstituents through selective media in the separation channel forsequential detection as the constituents elute into the detectionchannel from the separation channel. The microscale dimensions of theassay system can provide rapid analyses using microscale, or nanoscale,sample volumes. However, resolution or sensitivity may not be adequatefor complex samples or dilute samples.

One approach to enhancing the resolution and sensitivity of capillaryelectrophoresis (CE) methods has been to pre-resolve and pre-concentratethe sample using Isotachophoresis (ITP) before CE separations. In ITP,the sample is loaded into a channel between a leading electrolyte (LE)having an electrophoretic mobility greater than the sample and atrailing electrolyte (TE) having electrophoretic mobility less than thesample. Under the influence of an electric field, analytes of interestcan migrate through the sample bolus to accumulate at the interface withthe LE and/or TE solutions. In this way, the analytes of interest can beseparated from certain other constituents of the sample and concentrateto more detectable levels. Samples can thus be concentrated and desaltedto provide improved injection material for further capillaryelectrophoresis separations resulting in highly sensitive detectionswith high resolution. For example, in “Tandem Isotachophoresis-ZoneElectrophoresis via Base-Mediated Destacking for Increased DetectionSensitivity in Microfluidic Systems”, by Vreeland, et al., Anal. Chem.(2003) ASAP Article, sample concentrated by ITP is further resolved anddetected by capillary zone electrophoresis (CZE). In Vreeland, thesample is subjected to ITP between a TE and an LE having electrophoreticmobilities controlled by the pH of Tris buffers. While ITP concentrationof analytes progresses, hydroxyl ions (—OH) are formed by hydrolysis atthe cathode end of the separation channel. Migration of the hydroxylions through the separation channel eventually neutralizes the Trisbuffers to remove the mobility differences between the LE and TEsolutions. The Tris neutralization converts the ITP separation mediainto a CZE separation media. The analytes can than be separated withhigher sensitivity and resolution than for standard CZE of the samesample due to the effective sample volume reduction and concentration ofanalytes resulting from the ITP assay step. The Vreeland method islimited to pH based ITP of compatible samples, can be time consuming dueto the neutralization step, and can be inconsistent due to variations inbuffer preparation or hydroxyl ion generation.

In another scheme to combine ITP with CE, analytes of interest migratein ITP mode until they reach an intersection with a CE separationchannel before switching the electric field to the separation channelfor capillary electrophoresis separation of the analytes. For example,in “Sample Pre-concentration by Isotachophoresis in MicrofluidicDevices”, by Wainright, et al., J. Chromat. A979 (2002), pp. 69-80,samples are pre-concentrated in a ITP channel until they reach anintersection with a CE channel. The intersection is monitoredmicroscopically by a photomultiplier tube (PMT) receiving light througha confocal lens focused on the intersection. Analytes entering theintersection can be detected, e.g., by fluorescence or light absorption,and the electric field manually switched to inject the analytes into theCE channel. Problems exist, however, in that the manual switching can beinconsistent, some analytes may not be detectable using a PMT, and PMTdetection at the microscale can be cumbersome and expensive.

In view of the above, a need exists for increased sensitivity,consistency, and resolution of capillary and microscale electrophoresismethods. It would be desirable to have systems that can automaticallyand consistently switch between electrophoretic modes. The presentinvention provides these and other features that will be apparent uponreview of the following.

SUMMARY OF THE INVENTION

The present invention provides, e.g., systems and methods toconsistently inject analytes into separation media based on a triggeringvoltage event. The analytes can be preconditioned and concentrated in achannel by isotachophoresis (ITP) stacking, followed by application ofthe stacked analytes to a separation channel segment, when a voltageevent is detected in the channel.

The methods of the invention can provide highly repeatable analyticalresults with high sensitivity, speed and resolution. The method caninclude, e.g., analyte injection by stacking one or more analytes in astacking channel segment, detecting a voltage potential in the channel,and applying the stacked analyte into a separation channel segment byapplying an electric field or a pressure differential along theseparation channel segment when a selected voltage event is detected.The channel can be a microscale channel, e.g., with intersecting orcommon channel segments making up a loading channel segment, a stackingchannel segment and/or a separation channel segment.

Stacking of analytes can take place in a stacking channel segmentwherein analytes of interest can be sandwiched between buffers selectedto focus the analytes into a concentrated band during ITP. Typicalinjected analytes include, e.g., proteins, nucleic acids, carbohydrates,glycoproteins, ions, and/or the like. The stacking channel segment canhave a trailing electrolyte and/or a leading electrolyte which havedifferent mobilities. For example, the leading electrolyte can have afaster mobility under the influence of an electric field than thetrailing electrolyte or analytes of interest. In many embodiments, thetrailing electrolyte and the leading electrolyte can differ in pH,viscosity, conductivity, size exclusion, ionic strength, ioncomposition, temperature, and/or other parameters that can affectrelative migration of the electrolytes. The trailing electrolyte can beadjusted to have a mobility less than analytes so that the analytesaccumulate at the trailing interface during ITP. Optionally, the leadingelectrolyte can be adjusted to have a mobility greater than the one ormore analytes so that they can accumulate at the leading interfaceduring ITP separations. By narrowly adjusting the migration rates oftrailing and leading electrolytes, analytes can be focused between theleading and trailing electrolytes while sample constituents not ofinterest migrate to other zones of the stacking channel segment. Thatis, the trailing electrolyte can be adjusted to have a mobility greaterthan one or more sample constituents not of interest, or the leadingelectrolyte can be adjusted to have a mobility less than one or moresample constituents not of interest so that they are not focused withthe analyte of interest between the electrolytes.

When the channel of the analyte injection method includes separatestacking and separation channel segments, switching from the stackingchannel to the separation channel segment can be by switching theelectric field from the stacking channel segment to the separationchannel segment, e.g., when the stacked analyte enters an intersectionof the stacking and separation channel segments. For example, applyingan electric field to the separation channel segment can includeswitching from a substantial lack of current in the separation channelsegment while an electric current flows in the stacking channel segmentto an electric current in the separation channel segment while electriccurrent in the stacking channel segment is shut off. Shutting off(substantial lack) of current in the channel segments can be byapplication of a float voltage to prevent current flow in the channelsegment or simply by provision of a high resistance in the channelsegment (e.g., allowing no significant electric current outlet from thechannel segment). Optionally, switching can be by exerting a pressuredifferential across the separation channel segment.

The separation channel segment in the injection methods can resolveanalytes from other analytes or sample constituents. Such resolution canallow the analytes of interest to be identified or quantitated.Separation channel segments can have selective conditions or separationmedia to affect migration of analytes and sample constituents. Forexample, the separation channel can contain a pH gradient, sizeselective media, ion exchange media, a viscosity enhancing media,hydrophobic media, and/or the like.

Analytes resolved in separation channel segments can be detected foridentification and/or quantitation. Detectors can be focused to monitoranalytes in the separation channel segment or to detect analytes as theyelute from the separation channel segment. Detecting analytes can be bymonitoring parameters associated with the analytes, such as, e.g.,conductivity, fluorescence, light absorbance, refractive index, and/orthe like.

Sample solutions can be loaded to channels of the methods by a varietyof techniques, e.g., to provide adequate sensitivity and speed. Forexample, when the loading channel does not hold enough sample analytefor the desired detection, multiple samples can be consecutively loadedand stacked before fusion of multiple stacks to provide an enhancedconcentration of analyte in a small volume. Stacking two or more samplesof the analytes can proceed by, e.g.: loading a first sample into aloading channel; applying an electric field across the sample, therebystacking the sample; loading a second sample into the loading channel;and applying an electric field across the stacked sample and the secondsample to stack the second sample and cause the two stacked samples tobecome focused together between trailing and leading electrolytes. Themultiple stacking technique can be facilitated by flowing the stackedfirst sample towards the loading channel to clear excess electrolyte anddepleted sample solution before loading the second sample. Another wayto concentrate sample analytes can be, e.g., by loading samples of theanalytes in a loading channel comprising a cross-section greater than astacking channel segment cross-section so that analytes from a largesample volume do not have to migrate as far to accumulate at a trailingor leading electrolyte interface.

Spacer electrolytes, having migration rates intermediate to the trailingelectrolyte and the leading electrolyte, can be loaded between samplesand/or stacked analytes to resolve the sample into two or more analytesof interest. In one embodiment, stacking comprises loading one or morespacer electrolytes having a mobility greater than the trailingelectrolyte and less than the leading electrolyte between two or moreanalyte sample segments. In another embodiment, one or more of the twoor more analyte sample segments is a previously stacked sample analyte,and the spacer electrolyte is inserted during a multi-stacking loadprocedure. The spacer electrolytes can be adjusted to provide a mobilitybetween mobilities of two or more of the analytes in order to resolvethe analytes in ITP. Such spacer electrolyte adjustments can be made byselecting an appropriate electrolyte pH, spacer electrolyteconstituents, spacer electrolyte viscosity, spacer electrolyteconductivity, and/or the like.

In some injection methods, electrolytes can be intelligently formulatedto provide ITP resolution of analytes for injection. For example, if thepK of an analyte is determined, e.g., from experiments or calculations,leading and trailing electrolytes can be adjusted to pH valuesbracketing the pK so that analyte intruding into the leading electrolytebecomes less charged and less mobile, and/or analyte intruding into thetrailing electrolyte becomes more charged and more mobile. Suchadjustments can enhance the selectivity and concentration of ITP beforeinjection of the stacked analyte.

The injection of stacked analyte into a separation channel segment canbe triggered by detection of a selected voltage event. Voltages can bemonitored at various locations in the channel and voltage events thatprecisely indicate preferred timing for injection can be determined. Forexample, detecting a voltage event can include monitoring a floatvoltage necessary to maintain a zero current flow (or other definedcurrent flow) condition in the separation channel segment. Typicalvoltage events used to trigger the start of a separation can include,e.g., a voltage peak, a voltage trough, a predesignated voltage,relative voltage, or a rate of voltage change, (for example a zero slopeobserved at the top of a voltage profile). The switch to inject stackedanalyte from ITP to the separation channel segment can be an automaticapplication of an electric field or pressure differential along thechannel segment when the voltage event is detected.

Systems of the invention for injection of analytes can provide automatedinjection of stacked analytes for reliable, consistent, and sensitiveanalyses. Analyte injection systems can include, e.g., an analytestacking in a channel, a voltage detector in electrical contact with thechannel and in communication with a controller so that the controllercan initiate a flow of electrical current in a separation segment of thechannel, or a pressure differential along the channel segment, when aselected voltage event is detected by the voltage detector. Typically,the channel is a microscale channel having a loading channel segment, astacking channel segment, and a separation channel segment.

A stacking channel segment in the system is usually configured forisotachophoresis procedures with a trailing electrolyte (TE) and/or aleading electrolyte (LE). The electrolytes can have different adjustablemobilities. For example, the electrolytes can have different pH values,viscosities, conductivities, size exclusion cut-offs, ionic strengths,ion compositions, or temperatures. Analytes for stacking in the channelcan include molecules, such as proteins, nucleic acids, carbohydrates,glycoproteins, derivatized molecules, ions, and the like. Electrolytescan be tailored to selectively stack analytes of interest whilerejecting other sample constituents. For example, the trailingelectrolyte can be formulated to have a mobility less than the mobilityof the analyte of interest and a mobility greater than a mobility of asample constituent not of interest, so that the analyte accumulates onthe front of the TE while the constituent falls away through the TE. TheLE can be formulated to have a mobility greater than the mobility of theanalyte of interest and a mobility less than a mobility of a sampleconstituent not of interest, so that the analyte can accumulate at theLE interface while the constituent migrates away in front of the LEinterface.

The separation channel segment of the system can contain conditions orselective media to resolve analytes and constituents that have beenstacked in the stacking column. For example, the separation column caninclude a pH gradient, size selective media, ion exchange media,hydrophobic media, viscosity enhancing media, and the like.

The controller can receive output from the voltage detector to initiatean injection when a selected voltage event is detected. The controllercan be, e.g., a logic device or a system operator. In some embodiments,the injection event can be a switch from the stacking channel ITPelectric field conditions to driving forces required to insert stackedanalyte into a separation channel segment. For example, the injectioncan be a switch from the ITP current flow to substantial elimination ofcurrent in the stacking channel segment when the voltage event isdetected, while a field or pressure is initiated in the separationchannel segment.

The channel segments of the system can include a loading channel segmentin fluid contact with the stacking channel segment. Various loadingschemes can be employed to meet the demands of particular analyses. Inone embodiment, the loading channel segment can have a cross-sectiongreater than a stacking channel segment cross-section so that a largervolume of sample analyte can accumulate in the stacking channel segmentin a shorter amount of time, i.e., the average analyte molecule has ashorter migration distance across a large cross-section loading channelsegment than with a long loading channel segment of the same volume. Inanother aspect of loading, a first stacked analyte sample can be pulledback toward the loading channel segment before loading a second samplein a multiple stacking scheme to increase the analyte concentration andsensitivity of an assay. The “pull back” can be accomplished, e.g., byproviding a pressure differential across the stacking channel segment tocause the first stacked sample to flow back toward the loading channelsegment. Loading channel segments can be filled from, e.g., wells on amicrofluidic chip, or by fluid handling systems, such as receivingsamples from microarrays through a collector tube (sipper).

Spacer electrolytes can be used in the system, e.g., to enhanceresolution between two or more analytes of interest. For example, aspacer electrolyte with a mobility between the mobilities of two or moreanalytes can be introduced between sample segments containing theanalytes in the stacking channel segment. Analytes slower than thespacer electrolyte can partition behind the spacer while faster analytescan partition in front of the spacer. In an alternate embodiment, thesample analyte can be combined with spacer electrolytes, e.g., topartition into separate analyte zones, e.g., under the influence oftransient or steady state conditions in ITP.

Systems of the invention can have voltage detectors in communicationwith controllers to detect and respond to voltage events in channels.Voltage detectors can detect voltages between two or more electriccontacts across segments of channels, or between contacts at anylocation in the channel and a voltage reference, such as a ground. Insome embodiments of the systems, the voltage detector monitors thevoltage in the separation channel segment while stacking progresses. Thevoltage of the separation channel segment during stacking can bemonitored at an intersection with the stacking channel segment oranywhere along the separation channel segment, e.g., when no substantialcurrent flows in the separation channel segment, such as when a floatvoltage is being applied to the separation channel segment by a floatvoltage regulator, where there is no electrical outlet from one end ofthe channel segment, or where the channel segment has a controllingswitch in the off position.

Controllers can automatically switch the system from stacking mode toseparation mode on detection of a selected voltage event to injectstacked analytes into the separation channel segment. The voltage eventcan be, e.g., a voltage peak, a selected voltage, a voltage trough, arelative voltage, a rate of voltage change, and/or the like. Theautomatic switch can be, e.g., flowing of electrical current in thechannel segment, a change in relative voltages across a channel segment,or application of a pressure differential along the channel segment toinduce migration of the stacked analytes along the separation channelsegment.

Analytes separated in the separation channel segments can be detected byanalyte detectors of the system to identify and/or quantitate analytesof interest. Analyte detectors can be configured to monitor analytes inthe separation channel segment, or analytes eluting from the separationchannel segment. The analyte detector can comprise a fluorometer, aspectrophotometer, a refractometer, a conductivity meter, and/or thelike.

The systems of the invention are well suited to microfluidicapplications. For example, the loading channel segments, stackingchannel segments, separation channel segments, detection chambers, andthe like, can be incorporated into a microfluidic chip. The microscaledimensions of microfluidic devices are compatible with many systems ofthe invention. Microfluidic systems known in the art can providevoltages, pressures, fluid handling, communications, and detectors,etc., useful in practicing the systems of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C are schematic diagrams of an isotachophoresis system.

FIGS. 2A and 2B are schematic diagrams of transient ITP concentrating ananalyte at an interface with a leading electrolyte.

FIGS. 3A 3C are schematic diagrams of transient ITP separation ofanalytes of interest and steady state ITP juxtaposition of the analytes.

FIGS. 4A to 4C are schematic diagrams of selective removal of sampleconstituents during ITP.

FIGS. 5A to 5C are schematic diagrams of exemplary sample solutionloading techniques.

FIGS. 6A to 6E are sequential schematic diagrams describing a techniqueof stacking multiple loads of sample analytes.

FIGS. 7A to 7C are schematic diagrams showing enhanced sample solutionvolume loading using a loading channel segment with a cross-sectiongreater than the cross section of the stacking channel segment.

FIGS. 8A to 8D are schematic diagrams of voltage event detection at acontact point in a stacking channel segment.

FIGS. 9A to 9D are schematic diagrams of analyte band skewing caused byflow through a skewing channel.

FIGS. 10A to 10E are schematic diagrams of sample constituent skewingand dispersion in skewing channel ITP while an analyte of interest bandremains focused.

FIGS. 11A to 11C are schematic diagrams of stacked analyte applicationto a separation channel segment.

FIGS. 12A and 12B are schematic diagrams of a microfluidic chip with acollector tube feeding sample solutions to a loading channel segment.

FIGS. 13A and 13B are schematic diagrams of an analyte injection systemwherein a stacking channel segment shares a common channel with aseparation channel segment.

FIGS. 14A to 14C are schematic diagrams of an analyte injection systemincorporating skewing channels in spiral and serpentine configurations.

FIG. 15A to 15C are schematic diagrams of a skewing channel with anincreased ratio of outside travel distance over inside travel distancethrough a turn.

FIGS. 16A to 16C are schematic diagrams of a skewing channel withskewing provided by providing a travel surface distance on one sidegreater than for the other side of the channel.

DETAILED DESCRIPTION

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein havemeanings commonly understood by those of ordinary skill in the art towhich the present invention belongs.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular methods orsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “aconstituent” can include a combination of two or more constituents;reference to “the analytes” can include one analyte, and the like.

Although many methods and materials similar, modified, or equivalent tothose described herein can be used in the practice of the presentinvention without undue experimentation, the preferred materials andmethods are described herein. In describing and claiming the presentinvention, the following terminology will be used in accordance with thedefinitions set out below.

The term “stacking”, as used herein, refers to accumulation of one ormore analytes at an interface with electrolytes having differentmobilities in an electric field, e.g., during isotachophoresis. Stackingof analytes can also occur at other zones created, e.g., by migrationmodifying constituents present in the assay sample under transient(dynamic) or steady state conditions.

In “selective isotachophoresis” an analyte of interest with a knownmobility can be separated from another sample constituent, such as,e.g., a sample constituent not of interest, e.g., by providing leadingelectrolyte and/or trailing electrolytes with mobilities intermediate tothe mobilities of the analyte and sample constituent.

The term “analyte”, as used herein, refers to constituents of a samplethat are detected by an analyte detector. An “analyte of interest”, asused herein, refers to an analyte for which detection and/orquantitation is desired in an assay.

The term “channel” , as used herein, refers to a conduit for flowingand/or retention of fluids in methods and systems of the invention.Channels can be, e.g., tubes, columns, capillaries, microfluidicchannels, and/or the like. A channel can include various channelsegments, e.g., in separate sections of the channel, that share sectionsof the channel, and/or that intersect with other segments of thechannel. Channel segments are generally functional sections of channel,such as, e.g., loading channel segments, stacking channel segments, andseparation channel segments.

A “skewing channel” in the invention can be a channel segment thatcauses skewing of sample constituents flowing in the channel. Forexample, the internal surface topography of a skewing channel can causebands or peaks to take on an oblique orientation relative to the channelaxis while passing through the skewing channel.

The term “mobility”, as used herein, refers to a rate of migration forcharged molecules, such as analytes or electrolytes, in a solution underthe influence of an electric field in a channel.

The term “float voltage”, as used herein, refers to a voltage requiredin a channel segment to substantially prevent flow of an electriccurrent through the segment or to establish a desired constant currentin the segment.

The term “microscale”, as used herein, refers to dimensions ranging fromabout 1000 μm to about 0.1 μm.

The invention relates to methods and systems for injection of analytesinto separation channels. Stacking sample analytes can provide higheranalyte concentrations in smaller injection volumes for electrophoreticseparations with improved assay sensitivity and resolution. Sensitivityand separations can be improved, in many cases, by stacking analytes inskewing channels before injection. Automated timing of injectionstriggered by detection of voltage events can improve the consistency ofresults between assay runs.

Methods and systems of the invention can be used to separate, identify,and/or quantify analytes with a high level of sensitivity andresolution. Analytes of the invention can be, e.g., charged molecules,such as, e.g., proteins, nucleic acids, carbohydrates, glycoproteins,ions, derivitized molecules, and/or the like.

Methods of Analyte Injection

Methods of the invention can provide precise injection timing of stackedanalyte into a separation channel for sensitive, repeatable, highresolution assays. Methods of the invention generally include, e.g.,loading a sample to a loading channel segment before isotachophoresis(ITP) in a stacking channel segment, detecting a voltage event thatindicates a stacked sample analyte is in position for injection,applying an electric field or pressure differential to apply the stackedsample analyte to a separation channel segment, and detecting separatedanalytes of interest. The ITP can include migration of the analytesthrough skewing channels. Detection signals can be evaluated todetermine the presence or quantity of the analytes.

Stacking Analytes of Interest

Analytes of interest can be stacked into a volume less than the originalanalyte sample by isotachophoresis (ITP). For example, a sample boluscan be loaded between two different buffer systems in a channel andexposed to an electric current to create a steady state of solute zonesmigrating in order of decreasing mobility. In the steady state, thezones can adopt the same concentration and migrate along the channel atthe same velocity as the leading electrolyte. Alternatively, a samplebolus can be loaded adjacent to an electrolyte and stacked in a dynamic(transient) condition at the interface for injection, e.g., withouthaving reached a steady state equilibrium between ITP electrolytes.

Stacking can be practiced, e.g., in channels of a microfluidic chipwherein a sample is loaded between channel regions of a trailingelectrolyte and a leading electrolyte. As shown in FIG. 1A, analytesample 10 can be loaded to loading channel segment 11 by a differentialpressure between vacuum wells 12 and sample well 13. When an electricfield is applied across stacking channel segment 14, current is carriedby high mobility (e.g., high charge to mass ratio) leading electrolytes15, intermediate mobility analytes 16, and low mobility trailingelectrolyte 17, as shown in FIG. 1B. As ITP proceeds, a steady state canbe established in which the volume of analyte 16 is reduced to the pointwhere the concentration of charged analyte 16 is equivalent to theconcentration leading electrolyte 15. In the steady state, the stackedanalyte solution migrates along stacking channel segment 14 at the samerate as the leading and trailing electrolytes, as shown in FIG. 1C, withthe electrolytes and charged analytes carrying the same amount ofelectric current per unit volume in the stacking channel segment.Factors, such as charge density and transient differential migrationrates of the analytes and electrolytes, tend to focus the analytes andelectrolytes into zones during ITP. Stacking channel segments of theinvention can be any size including microscale channels having adimension, such as width or depth, ranging from about 1000 μm to about0.1 μm, or from about 100 μm to about 1 μm, or about 10 μm.

Stacking can also be practiced in a transient state. For example, asshown in FIG. 2A, initially dilute and dispersed analyte molecules 20can accumulate, e.g., at leading electrolyte interface 21 as shown inFIG. 2B. This concentration of analyte at an interface can occur beforeestablishment of steady state uniform analyte and electrolyte carrierconcentrations. Optionally, an analyte can accumulate in a transientstate, e.g., during initial application of an electric field in ITP, attrailing electrolyte interface 22. In other embodiments or transientITP, analytes can become concentrated in zones other than interfaces ofITP electrolytes.

Multiple analytes of interest can accumulate in a steady state ortransient state, e.g., at one or both of the electrolyte interfaces. Forexample, as shown in FIGS. 3A to 3C, sample solution 30 with firstanalyte of interest 31 and second analyte of interest 32 can be loadedbetween trailing electrolyte solution 33 and leading electrolytesolution 34. In the case where the first analyte has a slower mobilitythan the second analyte, but a faster mobility than the trailingelectrolyte, the first analyte can accumulate at the interface with thetrailing electrolyte in the presence of an electric field. Meanwhile, inthe transient state, as shown in FIG. 3B, the second analyte, withsomewhat higher mobility than the first analyte, can accumulate at theother end of the sample bolus along the interface with the fastermobility leading electrolyte. Such a situation can provide theopportunity for separate sequential or parallel application of the firstand second analytes to one or more separation channel segments, as canbe appreciated by those skilled in the art. Once a steady state has beenestablished in the ITP, as shown in FIG. 3C, charged first and secondanalytes can become compressed into narrow adjacent bands, e.g., forapplication together for resolution in a separation channel segment.

In methods of the invention, the mobilities of trailing electrolytes andleading electrolytes can be adjusted to provide selectivepre-concentration of an analyte of interest while separating sampleconstituents not of interest from the analyte. For example, as shown inFIG. 4A, sample solution 40 containing analyte of interest 41, slowmobility sample constituent not of interest 42, and fast mobility sampleconstituent not of interest 43, can be loaded between trailingelectrolyte 44 and leading electrolyte 45. When an electric field isapplied to the channel, slow mobility sample constituents not ofinterest 42 can fall behind the trailing electrolytes while fastmobility sample constituent not of interest 43 can race ahead of theleading electrolytes, as shown in FIG. 4B. Continued ITP to a steadystate can, e.g., further separate sample constituents not of interestfrom the analyte, as shown in FIG. 4C. Removal of sample constituentsnot of interest from analytes of interest can provide an improvedinjection material for separation in a separation channel segment. Aftersamples have been pretreated by ITP to remove sample constituents not ofinterest, analyses of analytes of interest applied to a separationchannel segment can have, e.g., reduced background noise, higherresolution due to lower injection volumes, more accurate quantitationsdue to better baselines and fewer overlapping peaks, etc.

Trailing electrolytes and leading electrolytes can be tailored,according to methods known in the art, by adjusting electrolytemobilities to provide highly specific retention and stacking of analytesof interest, while sample constituents not of interest are removed. Inone embodiment of the methods, the pH of electrolytes is selected tobracket the pK of an analyte of interest so that sample constituents notof interest having pKs outside the bracket will be removed in the ITP.The pK of the analytes of interest can be determined, e.g., empiricallyor based on the known molecular structure of the analytes. In otherembodiments, the analyte of interest can be, e.g., closely bracketedbetween selected trailing and leading electrolyte compositions known tohave slower and faster mobilities than the analyte. Many ions andbuffers can be used in electrolytes to bracket analytes, such as, e.g.,chloride, TAPS, MOPS, and HEPES. Optionally, the mobility ofelectrolytes and/or analytes can be modulated by adjusting the viscosityor size exclusion characteristics of the sample solution, trailingelectrolyte solution, and/or leading electrolyte solution. In anotheroption for adjusting the mobility of ITP solutions, mobility of analytesolutions and/or electrolyte solutions can be moderated, particularlyduring transient ITP migrations, by adjusting the concentration, ionicstrength, or conductivity of the solutions. The temperature of solutionscan be selected in still other options to adjust the mobility ofanalytes, electrolytes, or ITP solutions.

A variety of sample solution loading methods can benefit analyses inmethods of the invention. Stacking channels can be loaded with singlesample solution loads, with multiple sample solution loads, and withspacer electrolyte between sample solution loads, as described in detailbelow.

Single sample loads can be loaded to sample loading channel segmentsaccording to techniques known in the art, e.g., as shown in FIGS. 5A to5C. Sample solution 50 can be applied to loading channel segment 51using, e.g., electroosmotic flow (EOF) or a differential pressure toflow the sample solution from sample well 52 through the loading channelsegment and out through waste channel 53 intersecting and offset alongthe loading channel segment, as shown in FIG. 5A. Alternately, Samplesolution 50 can be loaded to branch into loading channel segment 51under the influence of a differential pressure between sample well 52and waste wells 54 as shown in FIG. 5B. In FIGS. 5A and 5B, thepressures in other wells with no flow must be adjusted to ensure zeroflows. In another sample loading alternative, a relative vacuum at wastewells 54 can draw sample solution 50, the trailing electrolyte, and theleading electrolyte in a “pinching” flow, as shown in FIG. 5C, forprecise and consistent definition of sample volumes.

Additional amounts of sample solution can be loaded for ITP using amultiple stacking technique. A first sample can be loaded into loadingchannel segment 60 as shown in FIG. 6A. An electric field can be appliedacross stacking channel segment 61 to stack sample analytes 62, as shownin FIG. 6B. The stacked sample analytes 62 can be flowed back towardsthe loading channel segment and second load of sample solution 63 loadedadjacent to the first stacked analytes, as shown in FIG. 6C. An electricfield can be applied across the stacking channel segment a second timeto stack the second sample analytes 64, as shown in FIG. 6D. Separationzone 65, substantially composed of trailing buffer, can exist initiallyduring the second stacking, but can dissipate as trailing electrolytesfall behind the second stack analytes in the electric field. Eventually,the first and second stacked analytes can combine under the influence ofthe electric field to form multiple stack 66 having, e.g., twice theamount of analytes as the first stack, as shown in FIG. 6E. The amountof analyte in the multiple stack can be further increased by additionalrounds of stack pull back, sample loading, and stacking.

Optionally, a large volume of sample solution can be loaded into aloading channel segment having a cross-section greater than thecross-section of the stacking channel segment. As shown in FIG. 7A,sample solution 70 can be loaded into large cross-section loadingchannel segment 71, e.g., with a differential pressure across samplewell 72 and waste well 73. Under the influence of an electric field,sample analytes 74 can be concentrated near the stacking channel segmententrance, as shown in FIG. 7B. Loading channel segments with increasedcross section can concentrate analytes in a shorter time due to thereduced axial distance 75 for analyte travel as compared to a similarvolume loading channel segment with a smaller cross section. Trailingelectrolyte 76 can optionally be brought to a position adjacent toconcentrated sample analytes 74 for subsequent ITP by, e.g., providing apressure differential to flush the loading channel segment with trailingelectrolyte, e.g., as shown in FIG. 7C.

Advantages can be obtained in methods of the invention by placing aspacer electrolyte between analyte sample segments for ITP. The spacerelectrolyte can have a mobility intermediate between the trailingelectrolyte and the leading electrolyte. The spacer electrolyte can havea mobility intermediate between two or more analytes of interest. Thespacer electrolyte can provide, e.g., enhanced resolution betweenmultiple analytes of interest. In one embodiment, spacer electrolyte canbe present in loaded sample solutions to provide a spacer zone betweenanalytes on application of an electric field. In another embodiment,spacer electrolyte can be loaded between cycles of multiple stacking.For example, multiple stacking can proceed as described above, but withspacer electrolyte present to the left of the initial stack, with spacerelectrolyte present in one or more loaded sample solution segments, orby loading spacer electrolyte between cycles of loading sample solutionsegments. Spacer electrolytes can be adjusted as described above foradjustment of trailing and leading electrolyte mobilities to tailorspacer migration between analytes of interest.

Detecting Voltage Events

Detection of voltage events associated with, e.g., migration ofsolutions, analytes, and/or electrolytes in the stacking channel segmentcan provide, e.g., a consistent signal for initiation of stacked analyteapplication to a separation channel segment. During an ITP, voltagepotentials across the stacking channel segment, or voltages measurableat any point along the stacking channel segment, can vary with time.From one ITP run to the next, there can be measurable voltage eventsthat are consistent between runs and which can act as timing markersuseful for consistent triggering of injections and the switch from anITP to a different separation scheme.

In a typical embodiment of detecting a voltage event, trailingelectrolyte, analyte, and leading electrolyte are flowing in a stackingchannel segment during an ITP. The trailing electrolyte has a higherresistance to electric current flow than the leading electrolyte. With avoltmeter monitoring voltage, e.g., at a point half way along thestacking channel segment, as shown in FIG. 8, voltage events can bedetected as the ITP proceeds. With sample solution 80 initially loadedand applied to the stacking column entrance, leading electrolyte 81fills the stacking channel segment and the voltage detected at contact82 half way along the channel segment is about half the ITP electricfield voltage. As the analyte and trailing electrolyte 83 migrate downthe stacking channel segment, resistance increases on the entrance sideof the stacking channel segment resulting in a detectable voltage riseat the voltmeter contact, as shown in FIG. 8B. At about the time stackedanalyte reaches the point of voltmeter contact, the difference inelectrical resistance on the two sides of the point of contact reaches amaximum along with the detected voltage, as shown in FIG. 8C. Finally,as the analyte approaches the end of the stacking channel segment, nowsubstantially filled with trailing electrolyte, the resistance on bothsides of the contact equalize and detected voltage returns to about halfthe ITP electric field voltage, as shown in FIG. 8D. Voltage events, inthis example could include the starting voltage value, the start ofvoltage rise, the rate of change (slope) of the voltage rise or fall,the maximum voltage (voltage peak), the slope of zero observed atmaximum voltage, the return to starting voltage, any predeterminedvoltage, any relative voltage between locations in the channel segments,and/or the like. Consistent, but somewhat different, voltage profilescan be observed, e.g., with one or more voltmeter contacts located atdifferent points along the stacking channel segment. These consistentmeasurable voltage events can be selected, e.g., to trigger switches inelectric current or pressure differentials in channel segments to applystacked analytes to a separation channel segment.

A separation channel segment in electrical contact with a stackingchannel segment will have no substantial flow of electric current if theseparation channel is not part of a complete circuit (e.g., a “dead end”with no ground connection) or if a float voltage is applied to theseparation channel segment. In a preferred configuration for detectingvoltage events, the voltmeter contact can be located at a point betweenthe separation channel segment and the stacking channel segment, or atany location along the separation channel segment. In one preferredembodiment, voltage events can be detected by monitoring a separationchannel segment float voltage.

Enhancing Separations in Skewing Channels

Separation of analytes of interest from other sample constituents can beenhanced by stacking the analyte during and/or after passage through askewing channel segment. For example, sensitivity of an assay can beincreased when sample constituents not of interest become dispersed bythe turns while the analyte of interest continues to be focused byelectrolytes in the isotachophoresis method.

Analyte bands flowing in channels of an analytical system can becomedispersed when the channel diverges from a straight path. For example,as shown in FIGS. 9A to 9D, analyte 90 flowing on the inside of turn 91travels a shorter distance than analyte flowing on the outside of theturn. The initially compact band can become skewed and dispersed along agreater length of the channel, as shown in FIG. 9C. Axial diffusion ofthe skewed band can dilute the band and prevent realignment of the band,as shown in FIG. 9D. A detector focused on the band in FIG. 9A woulddetect a stronger and narrower maximum signal for the band than adetector focused on the band in FIG. 9D after skewing and diffusion.Such dispersion of bands can be problematic an many chromatographicanalysis because of the resultant broadening and shortening of peaks.However, the present invention can combine, e.g., intentionallyaccentuated skewing with ITP technology to enhance separations bystacking analytes of interest while dispersing sample constituents notof interest.

In one embodiment, for example, a small amount of analyte of interestcan be separated from a larger amount of sample constituent not ofinterest with an enhanced degree of sensitivity and improvedquantitation. In an ITP system without skewing channels, as shownschematically for example in FIG. 10A, a small amount of stackinganalyte of interest 100 can migrate, e.g., between selected trailing andleading electrolytes, while a larger amount of sample constituent not ofinterest 101, with a mobility similar to the trailing electrolyte,migrates near the front of the trailing electrolyte. Detector 102focused on the channel can fail to resolve the analyte and sampleconstituent peaks, as shown in detector output signal chart 103. Theanalyte sensitivity and quantitation capabilities can be enhanced by,e.g., introducing one of more skewing channel segments into the stackingchannel. Analyte 100 and sample constituent 101 migrating in the stakingchannel (FIG. 10B) can become skewed and dispersed in skewing channel104 (FIG. 10C). Some time after exiting the skewing channel, the stakingforces of the leading and trailing electrolytes can focus and realignthe analyte peak in the channel, while the un-shepherded sampleconstituent peak remains skewed and becomes diffused. A detector focusedon the channel can detect the presence and quantity of analyte against adiminished and less intrusive background of sample constituent.

The benefits of ITP separations in skewing channels can be increased byselecting trailing and/or leading electrolytes to enhance the stackingfocus of the analyte while increasing the mobility difference betweenthe electrolytes and the sample constituent. In selective ITP themobilities of leading and trailing electrolytes are selected, e.g., tobe near the known mobility of an analyte and/or to increase thedifference in mobility between the electrolytes and one or more sampleconstituents not of interest. For example, in the situation describedabove, where the analyte of interest has a mobility greater than thesample constituent not of interest, the trailing electrolyte can beselected to have a mobility closer to the analyte than to the sampleconstituent so that, e.g., the analyte is closely shepherded while thesample constituent lags behind to experience the effects of skewing anddiffusion. In a similar fashion, if the analyte of interest has amobility less than the sample constituent not of interest, the mobilityof the leading electrolyte can be selected to be between the analytemobility and the sample constituent mobility to enhance skewing channelITP separation. In a preferred embodiment, the mobility of anelectrolyte is selected to be between the mobilities of the analyte ofinterest and one or more sample constituents not of interest but closerto the mobility of the analyte. In another example, both leading andtrailing electrolytes can be selected to be close to the known mobilityof the analyte of interest. This can provide particular benefits whenboth faster and slower sample constituents migrate near the analyteand/or when transient stacking prevails during the ITP.

The effectiveness of skewing channel ITP can vary widely depending onfactors, such as, e.g., the radius of any turns involved, the internaldiameter of the channel, the topography of the channel walls, the crosssection of the skewing channel, the flow velocity, and the viscosity ofsolutions. For example, as is discussed in the Skewing Channel ITPSystems section below, skewing in a channel can be increased with shortturn radii, repeated turns in the same direction, channel topographiesthat increase the difference between the surface length of oppositechannel walls, and channel cross sections that are wider perpendicularto the axis of a turn. Appropriate conditions for a particular method orsystem can be derived, e.g., through calculation and/or experimentation.

To consider how diffusion can affect the amount of skew caused by aturn, a two-dimensional, nondimensionalized advection-diffusion equationcan be considered (see also, Analytical Chemistry, vol 73, No. 6,1350-1360, Mar. 15, 2001):${\frac{\partial c^{\prime}}{\partial t^{\prime}} + \underset{\underset{advection}{︸}}{u^{\prime}\frac{\partial c^{\prime}}{\partial x^{\prime}}}} = {\frac{1}{{Pe}_{w}^{\prime}}\left( {\underset{\underset{\underset{diffusion}{axial}}{︸}}{\frac{w}{L}\left( \frac{\partial^{2}c^{\prime}}{\partial x^{\prime 2}} \right)} + \underset{\underset{\underset{diffusion}{transverse}}{︸}}{\frac{L}{w}\left( \frac{\partial^{2}c^{\prime}}{\partial y^{\prime 2}} \right)}} \right)}$wherein L is the length of the turning channel, w is the internal widthof the turning channel, and Pe′_(w) is the dispersion Peclet number; u′,c′, t′, x′ and y′ are the normalized velocity, concentration, time,axial channel dimension, and transverse channel dimension, respectively.Three parameters, Pe′_(w), L, and w, have been determined to be ofspecial importance to skewing and dispersion of analytes under theinfluence of skewing channels in the present invention.

The Peclet number (Pe) is a dimensionless factor representing a ratio ofadvection (or forward movement) and diffusion of an analyte. If Pe islarge, peaks skewed by passage through a first skewing channel canretain a stable oblique shape long enough to have it reversed by asecond turn in the opposite direction. If Pe is small, peaks skewed in askewing channel can diffuse across the width of the channel in arelatively short time to convert a skewed peak into a diffuselybroadened peak. In methods of the invention, sample constituents not ofinterest can be most readily skewed and dispersed from analytes ofinterest, e.g., when conditions exist in skewing channels providing aPeclet number more than about the ratio of the length of the skewingchannel over the internal width of the skewing channel (i.e., Pe>L/w).Significant benefits in skewing, diffusion, and dispersion of sampleconstituents not of interest in skewing channel ITP can be obtainedwhere conditions provide a Peclet number more than about 0.01 times, 0.1times, 1 time, 10 times, 100 times, or more, than the ratio of theskewing channel length over the skewing channel width.

Conditions affecting the Peclet number can be, e.g., conditions thatinfluence advection and/or diffusion of molecules in the channels, as isknown by those skilled in the art. For example, Pe can be influenced bythe viscosity of solutions, the presence of a gel, temperature,molecular concentrations, the velocity of the molecule along thechannel, the diameter of the channel, and/or the like. Adjustment ofconditions controlling advection and diffusion can provide Pecletnumbers, e.g., that result in desirable levels of sample constituentdispersion during and/or after passage through skewing channel segmentsof the invention.

Applying Stacked Analytes to Separation Channels

Analytes stacked by ITP can be injected into a separation channelsegment, e.g., by applying an electric field or pressure differentialacross the separation channel segment and the stacked analytes. Thefield and/or pressure can cause migration or flow of analytes into theseparation channel segment. Application of the field or pressure can betriggered by detection of a voltage event, as described above, toprovide consistent and functional analyte injection timing. Applicationof the separation channel segment electric field or pressuredifferential can coincide with elimination of current flow in thestacking channel segment. The timing between the voltage event and theinjection can be established to conform to particular configurations ofchannels, intersections, and solution segments. The timing can also playa key role in determining the peak resolution and signal strength as itcan affect the amount of transient isotachophoresis that persists afterthe handoff.

Separation channel segments can provide conditions for electrophoreticseparation of analytes and/or separation by selective media. Inpreferred embodiments, separation channel segments have a microscaledimension (e.g., a depth or width ranging from about 1000 μm to about0.1 μm, or from about 100 μm to about 1 μm), e.g., to provide fastseparations of small analyte sample volumes. Separation channel segmentscan have separation media, such as, e.g., a pH gradient, size selectivemedia, ion exchange media, a viscosity enhancing media, hydrophobicmedia, and/or the like, capable of contributing to the resolution ofanalytes. Separation channel segments (as well as stacking channelsegments) can have viscosity enhancing media, such as gels, to reduceelectroosmotic flow (EOF) in separation modes where EOF is undesirable.Separation channel segments can be independent from other channelsegments, or can share all or part of a channel with other channelsegments, such as, e.g., loading channel segments and stacking channelsegments. In a preferred embodiment, the separation channel segment isindependent, but intersects in a fluid contact at some point along thelength of the stacking channel segment.

In a typical embodiment, stacked analyte from an ITP separation isinjected into a separation channel segment when a peak voltage isdetected at the intersection of a stacking channel segment and theseparation channel segment. For example, the float voltage in separationchannel segment 110 reaches a maximum (and the rate of voltage change,or slope of the voltage profile, becomes zero) as stacked analytes 111,sandwiched between trailing electrolyte 112 and leading electrolyte 113,migrate in an ITP past a voltmeter contact at the intersection of theseparation channel segment with the stacking channel segment, as shownin FIG. 11A. The voltage maximum can trigger the elimination of the ITPelectric field in the stacking channel segment and the application of anelectrophoresis electric field in the separation channel segment toinduce migration (application) of stacked analytes 111 into theseparation channel segment, as shown in FIG. 11B. Migration of analytesthrough selective media of the separation channel segment can separate(resolve) analytes of interest 114 from sample constituents not ofinterest 115 that co-migrated with the analytes through the stackingchannel segment during ITP, as shown in FIG. 11C. In some embodiments,multiple analytes of interest that stacked together, or in proximity toeach other, during ITP can be resolved from each other in the separationchannel segment, e.g., by capillary zone electrophoresis.

Alternate schemes for timing of injection will be appreciated by thoseskilled in the art. Such alternate schemes can be based, e.g., oncalculations or models, or can be determined empirically. For example,time delays can be built into triggered responses based on channelvolumes, channel geometry, voltmeter contact location, choice of voltageevents, the location of analytes relative to solution features affectingvoltage events, and/or the like. In a particular example, whereinanalyte is stacked near a trailing electrolyte interface in a transientITP (not yet reaching a steady state) and the remaining sample solutionbolus has a high electrical resistance, a suitable trigger time can be acertain time after the voltage peak to allow the stacked analyteadditional migration time to reach the intersection with the separationchannel segment.

Application of an electric field along the separation channel segmentcan be automatic (that is, not requiring manual switching). Suchautomatic application of the electric field can be accomplished, e.g.,by electronic devices and algorithms known in the art. For example, avoltmeter can be set to trip a switch when voltage at a contact reachesa set level. In preferred embodiments, a logic device, such as, e.g., anintegrated circuit or a computer, can be programmed to initiateswitching of actuators according to preset parameters (e.g., theoccurrence of defined voltage events).

Detecting Analytes

Analytes separated in by methods of the invention can be detected in theseparation channel segment and/or sequentially after elution from theseparation channel segment. Appropriate detectors can, e.g., be fixed tomonitor analytes in a detection channel, sequentially scan for analytesin channel segments, or provide continuous imaging of entire channels.

Appropriate detectors are often determined by the type of analyte to bedetected. Proteins and nucleic acids, for example, can often be detectedby spectrophotometric monitoring of particular light absorptionwavelengths. Many ionic analytes of interest can be detected bymonitoring changes in solution conductivity. Many analytes arefluorescent or can be labeled with fluorescent markers for detectionusing a fluorometer. Many analytes in solution, particularlycarbohydrates, can be detected by refractometry.

In a typical embodiment, detecting can be by monitoring transmission ofa light source through a separation channel segment using aphotomultiplier tube (PMT) focused on the channel with a microscopelens. Those skilled in the art will appreciate how such an arrangementcan be configured as a fluorescence detector by addition of anappropriate excitation light source, such as, e.g., a laser or filteredlight from a lamp. Optionally, the lens can be mounted on an X-Yscanning mechanism to monitor any location on a microfluidic chip. Withsuch an arrangement, the length of a separation channel segment can bescanned for analytes, e.g., resolved along a pH gradient. In anotherembodiment, conductivity meter sensors can be mounted across aseparation channel outlet to monitor charged analytes as they elute fromthe channel segment.

Detectors can be in communication with data storage devices and/or logicdevices to document assay runs. Analog output from detectors, such asPMTs and conductivity meters, can be fed to chart plotters to retain atrace of the analyte separation profile on paper. Analog to digitalconverters can communicate detection signals to logic devices for datastorage, separation profile presentation, and/or assay evaluation.Digital logic devices can greatly facilitate quantitation of analytes bycomparison to appropriate standard curves from regression analysis.

Analyte Injection Systems

Electrokinetic analyte injection systems described herein can providesensitive analyte detection with high resolution in a highly consistentmanner. Analytes selectively stacked in a stacking channel segment canbe injected (applied) into a separation channel segment with precisetiming based on detection of voltage events in the channels. Suchprecision can be enhanced by provision of automated injectionsubsystems.

Systems of the invention generally include, e.g., an analyte stacking ina channel, a voltage detector in communication with a controller and incontact with the channel at one or more locations, an electric currentor pressure differential established in the channel when a selectedvoltage event is detected by the voltage detector and communicated tothe controller. The channel can include stacking channel segments andseparation channel segments that intersect, form a continuous channel orwhich share common channel sections. Analytes applied to the separationchannel segment and separated can be, e.g., detected by a detector incommunication with a logic device to determine the presence ofparticular analytes or to evaluate the quantity of analytes.

Channels

The channel of the invention can be, e.g., a single multifunctionchannel comprising loading segments, stacking segments, separationsegments, and/or detection segments. Optionally, the channel can includeseparate loading channel segments, stacking channel segments, andseparation channel segments in fluid contact at intersections. In apreferred embodiment, as shown schematically in FIG. 11, the loadingchannel segment is an extension of the stacking channel segment, and theseparation channel segment is in fluid contact with the stacking channelsegment through an intersection where analyte injection takes place.Channels of the systems can be any known in the art, such as, e.g.,tubes, columns, capillaries, microfluidic channels, and/or the like. Ina preferred embodiment, the channels are microscale channels, e.g., on amicrofluidic chip.

Channels of a microfluidic device can be embedded on the surface of asubstrate by mold injection, photolithography, etching, laser ablation,and the like. The channels can have a microscale dimension, such as,e.g., a depth or width ranging from about 1000 μm to about 0.1 μm, orfrom about 100 μm to about 1 μm. Fluids can flow in the channels, e.g.,by electroosmotic flow, capillary action (surface tension), pressuredifferentials, gravity, and/or the like. Channels can terminate, e.g.,in wells of solutions and/or at intersections with other channels orchambers. Channels can have electrical contacts, e.g., at each end toprovide electric fields and/or electric currents to separate analytes orto induce EOF. Detectors can be functionally associated with channels tomonitor parameters of interest, such as, e.g., voltages, conductivity,resistance, capacitance, electric currents, refractivity, lightabsorbance, fluorescence, pressures, flow rates, and/or the like.Microfluidic chips can have functional information communicationconnections and utility connections to supporting instrumentation, suchas electric power connections, vacuum sources, pneumatic pressuresources, hydraulic pressure sources, analog and digital communicationlines, optic fibers, etc.

The channel can include, e.g., a load channel segment to introduce oneor more sample solution volumes into the channel. Such loading channelscan be configured in ways appreciated by those skilled in the art, suchas, e.g., as an injector loop, to include an a collector tube 120 to amicrofluidic chip, as shown in FIG. 12, and/or as a flushed channelsegment, as shown schematically in FIGS. 5A to 5C. Loading channelsegments can have a cross-section greater than the cross-section of thestacking channel segment, as shown in FIG. 7, to provide rapidconcentration of analytes near the stacking channel segment entrancefrom a large volume of sample solution.

Channels of the systems can contain gelatinous substances tobeneficially affect migration and flow characteristics of the channels.Gels can be incorporated into channels to reduce unwanted electroosmoticflows of solutions while providing a more electrophoretic character to aseparation. Gels can influence the relative migration rates of analytesand/or electrolytes by slowing the progress of larger molecules. Gelscan provide tools to help adjust migration zones for analytes and ITPelectrolytes in stacking channel segments. For example, analytes ofinterest are generally larger than commonly used ITP electrolytes. Byplacing a gel in a stacking channel segment, a fast analyte (large butwith a high charge to mass ratio) can be slowed to migrate behind aleading electrolyte small molecule salt or buffer. Optionally, a gel canslow an analyte to migrate only marginally faster than a trailingelectrolyte. Gel resistance to large molecule migration can beadjustable, e.g., by altering the type of gel, concentration of gelmatrix, and the extent of gel matrix cross-linking. Gels can provideenhanced concentration and/or resolution to analytes in stacking orseparation channel segments. One or more different gels can be presentin either the stacking channel segment or the separation channelsegment.

Stacking channel segments can function to selectively stack analytes ofinterest, e.g., by ITP, for injection into a separation channel segmentfor further resolution and detection. Stacking channel segments can haveelectrical contacts, e.g., at each end, for application of electricfields suitable for analyte stacking. Stacking channel segments can havefluid contacts with, e.g., externally driven pneumatic or hydraulicmanifolds so that pressure driven flows, such as electrolyte loading orthe pull back for the multiple stacking technique discussed in the“Stacking Analytes of Interest” section above, can be practiced.Stacking channel segments can contain, e.g., electrolytes, such astrailing electrolytes, spacer electrolytes, and/or leading electrolytes,suitable for isotachophoresis (ITP), as discussed in the Methods sectionabove. The stacking channel segment can have trailing electrolyte well18, as shown in FIG. 1, and leading electrolyte well 19, forintroduction of electrolytes into channel segments.

Separation channel segments can receive stacked analytes by injectionfrom stacking channel segments for further resolution by separationtechniques, such as, e.g., additional rounds of ITP, ion exchange, sizeexclusion, hydrophobic interaction, reverse phase chromatography,isoelectric focusing, capillary zone electrophoresis, and/or the like.Separation channel segments can include electric contacts forapplication of electric fields along the channel segment and/or externalconnections with pressure sources to drive fluid flows. Separationchannel segments can be, e.g., a channel segment intersecting a stackingchannel segment, a channel segment continuing a common channel with astacking channel segment, and/or a channel segment functionally sharingchannel sections with a stacking channel segment. In a typicalembodiment, the separation channel segment intersects the stackingchannel segment at some point along the stacking channel segment length,as shown in FIG. 11. In this embodiment, sample constituents not ofinterest can remain in separate stacking channel segment sections afterinjection of stacked analytes of interest into the separation channelsegment. In other embodiments, e.g., the stacking and separation channelsegments can functionally reside in a common channel without anintervening intersection. For example, e.g., as shown in FIG. 13A,stacking can continue in a channel segment until a voltage event isdetected. On detection of the voltage event, conditions can change inthe channel for a transition to a separation mode. Such a transition caninclude, e.g., application of a differential pressure between channelends 130 to induce analyte flow into size exclusion resin 131, as shownin FIG. 13B. Smaller molecules will elute past detector 132 beforelarger molecules. Other examples of transitions to separation modes caninclude, e.g., changes in the direction of electric current flow,changes in the direction of fluid flow, injections of separation buffersinto a channel, changes in an electric field voltage, and/or the like.

Skewing Channel ITP Systems

Isotachophoresis systems of the invention can include skewing channelsegments in, and/or before, the stacking channel to enhance theseparation of analytes of interest from sample constituents not ofinterest. The sample constituents can be dispersed while the analyte ofinterest is focused by stacking, e.g., in the skewing channels. Theseparation enhancement can be promoted, e.g., by turning throughcumulatively large angles, sharp turning, skewing channel cross sectionshaving relatively large widths, skewing channel topographies withopposite surfaces of different length, and/or skewing channel systemshaving conditions providing a Peclet number more than about the ratio ofthe skewing channel length over the skewing channel width.

One way to increase skew and dispersion in skewing channel segments isto provide greater turning angles in the channel. In a two dimensionalplane, turning angles can be accumulated, e.g., with continuous spiralturns or switching serpentine turns as shown in FIGS. 14A to 14C. Spiralturns have the advantage that turning angles can accumulate through alarge number of degrees in one direction, with a concomitantaccumulation of skew. A disadvantage of spiral skewing channels can bethe inherent continuous expansion of the turn radius into a range ofless effective curvatures. Spiral skewing channel configurations canalso entail difficult access problems for connections to the innerchannel end. One way to provide accessible channel ends in a spiralskewing channel configuration can be to have side by side spiralingchannels running in and out of the center, as shown in FIG. 14B.Alternately, the access to a spiral channel end can be provided in thethird dimension, e.g., through a sipper tube or a back channel inanother plane, e.g., as shown in FIG. 14A. Another limitation on thelength of the spiral channel is that the Peclet number required foroptimal skewing increases as the length of the spiral channel increases.Serpentine skewing channels, as shown in FIG. 14C, can provide easyaccess to channel ends but complimentary turns can cancel the skew ofprevious turns, particularly where the Peclet number is large or thetime is short between turns. Optionally, three dimensional skewingchannels can be employed, such as helices and coils.

Skew and dispersion from passage through skewing channel segments can bemore pronounced in channels that make sharp turns relative to theinternal channel diameter. For example, skew is increased for skewingchannels with a high ratio of channel internal diameter over turn width.In one embodiment, the skew from a skewing channel segment having turnsis increased when the cross-section of the channel is greater along theradius of the turn (skewing channel internal width) than perpendicularto the turn radius (skewing channel depth).

The topography of a skewing channel segment can affect the skew anddispersion of migrating analytes. For example, channel surface contoursthat increase the ratio between the travel distance along the outside ofa turn over the travel distance along the inside of the turn canincrease skew. Skew can be increased by increasing channel internalwidth relative to channel depth at turn points. As shown in FIG. 15,analyte 150 can become highly skewed by flowing through a turn having abolbus outer turn surface. Skew can be enhanced in skewing channelswhere the travel surface distance on a first side 151 of the skewingchannel is greater than the travel surface distance on a second side 152of the skewing channel, even if there is no curvature in the skewingchannel overall, as shown in FIG. 16. For example, significant skewingcan be provided from differences in opposite surface travel distancesranging from more than about 500%, to 100%, to 50%, to 10%, or less.

Selective stacking of the analyte of interest between leading andtrailing electrolytes is an important aspect of skewing channel ITPsystems of the invention. Analytes of interest can be continuouslyrefocused between the electrolytes during and/or after skewing whilesample constituents not of interest become dispersed. The mobility of ananalyte of interest can be known from calculations or by empirical data.The trailing and/or leading electrolyte can be selected to have amobility between those of the analyte of interest and intrusive sampleconstituents not of interest. To enhance focusing of the analyte anddispersion of sample constituents, the electrolytes can be selected tohave mobilities closer to that of the analyte of interest than thesample constituents.

Skewing ITP channel segments can be incorporated into the systems andmethods of injecting analytes described above. An analyte of interestcan be injected into a separation channel at higher purity afterdispersion of other sample constituents by skewing channel ITP.Injection of the analyte can be initiated on detection of a voltageevent.

Voltage Detectors

Voltage detectors in systems of the invention can be in contact withchannels to detect voltage events communicated to a controller. The typeand complexity of voltage detectors can depend on, e.g., channelhardware configurations and the type of voltage event to be detected.

Voltage detectors can range from, e.g., simple relay switches tripped bya voltage, to analog galvanometers, to analog devices with chartrecorders, to voltmeters with digital outputs for evaluation by logicdevices. Voltmeters generally detect a voltage potential betweenelectrodes at two locations, such as, e.g., a contact location in achannel and a ground, or between two different locations in a channel.The location of the voltage electrode contacts with the channel canchange the voltage profile detected during a stacking run. However, awell defined voltage event can often be determined for consistent andunambiguous triggering of an injection for voltmeter contacts at a widerange of channel locations (e.g., the voltmeter contact does not have tobe at an intersection between stacking and separation channel segments).

In one embodiment, voltmeter contacts can be located at two ends of thechannel. As trailing electrolyte, of relatively high resistance,displaces leading electrolyte in the channel, the voltage required tomaintain a selected current through the channel can increase. A voltageevent to trigger injection in this case can be, e.g., a preset voltage.

In another embodiment, voltmeter contacts can be located at a ground (orother voltage reference) and at any point in a separation channelsegment intersecting a stacking channel segment. If electric current isnot allowed to flow through the separation channel segment (e.g., wherethe separation channel segment is held at zero current by a floatvoltage, or where the separation channel segment not part of a completecircuit), any location in the separation channel segment will reflectthe stacking channel segment voltage at the intersection. Voltagedetected in the separation channel segment can rise to a peak and fallas the TE/LE interface passes the intersection, in a fashion similar tothe voltage profile of FIG. 8, as will be appreciated by those skilledin the art.

Where voltage is being monitored in a separation channel segment withoutelectrical current and in contact with the stacking channel segment, thelack of current can be by, e.g., float voltage regulation or circuitisolation. A float voltage regulator device can be an electronic device,known in the art, that detects electric current flow in a channelsegment and applies a voltage to the channel segment that neutralizesany voltage potential across the channel segment, thus preventing a flowof electric current. A float voltage regulator can optionally beconfigured to adjust a channel segment voltage differential to provide aselected constant current in the channel segment. Another way to preventelectric current flow in a channel segment is to ensure that the channelsegment is not a part of a completed electric circuit. For example, anelectric switch can be present at one end of the channel segment toselectively open or close any associated electric circuits.

The voltmeter can communicate with a controller for initiation ofanalyte application (injection) to a separation channel segment.Initiation of injection can be manual or automatic. For example, thevoltmeter can provide a visible voltage readout for a system operator(the controller) to manually switch channel electric fields or fluidflows on observation of a voltage event, such as a selected voltage orvoltage peak. In another example, the controller is a digital logicdevice in electronic communication with the voltmeter and set toautomatically apply stacked analytes to a separation channel segment ondetection of a selected voltage event.

Analyte Detectors

Appropriate analyte detectors can be incorporated into systems of theinvention to detect analytes. The type and configuration of detectorscan depend, e.g., on the type of analyte to be detected and/or on thelayout of channels. Analyte detectors can be in communication with logicdevices for storage of analyte detection profiles and evaluation ofanalytical results.

Analytes for detection in the systems can range widely, with many beingcharged molecules or molecules modified to have a charge. For example,analytes of interest can be proteins, nucleic acids, carbohydrates,glycoproteins, ions, and/or the like. Although stacking can take placeby alternate mechanisms, such as size exclusion, stacking is driven bymigration of charged analytes in an electric field for many systems ofthe invention. It will be appreciated by those skilled in the art thatnon charged analytes of interest can receive a charge forelectrophoretic stacking by appropriate adjustment of pH orderivatization of the analyte with a charged chemical group.

Analyte detectors in the systems can be any suitable detectors known inthe art. For example, the detectors can be fluorometers,spectrophotometers, refractometers, conductivity meters, and/or thelike. Analytes not detectable by available detectors can often bederivitized with a marker molecule to render then detectable. Thedetectors can be mounted or focused to monitor analytes in the channelsegments, including, e.g., intersections and/or separation channelsegments. Detectors can monitor analytes as they exit separation channelsegments, e.g., in detection channels of chambers.

Analyte detectors can monitor a channel location, sequentially scan achannel length, or provide a continuous image of separated analytes. Inone embodiment, a stationary spectrophotometric detector can be aphotomultiplier tube focused on a particular channel location orintersection. In another embodiment, the analyte detector can be afluorometer focused on microchannels through a confocal microscope lensmounted to an X-Y transporter mechanism to sequentially scan analytesseparated in channels of a microfluidic device. In another embodiment,the analyte detector can be a charge coupled device (CCD) array capableof providing an image of numerous separations in multiple separationchambers at once.

The analyte detector can be in communication with a logic device forstorage and evaluation of analytical results. Logic devices of thesystems can include, e.g., chart recorders, transistors, circuit boards,integrated circuits, central processing units, computer monitors,computer systems, computer networks, and/or the like. Computer systemscan include, e.g., digital computer hardware with data sets andinstruction sets entered into a software system. The computer can be incommunication with the detector for evaluation of the presence,identity, quantity, and/or location of an analyte. The computer can be,e.g., a PC (Intel x86 or Pentium chip—compatible with DOS®, OS2®,WINDOWS® operating systems) a MACINTOSH®, Power PC, or SUN® work station(compatible with a LINUX or UNIX operating system) or other commerciallyavailable computer which is known to one of skill. Software forinterpretation of sensor signals or to monitor detection signals isavailable, or can easily be constructed by one of skill using a standardprogramming language such as Visualbasic, Fortran, Basic, Java, or thelike. A computer logic system can, e.g., receive input from systemoperators designating sample identifications and initiating analysis,command robotic systems to transfer the samples to the loading channelsegments of the system, control fluid handling systems, control detectormonitoring, receive detector signals, prepare regression curves fromstandard sample results, determine analyte quantity, and/or storeanalytical results.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, many of the techniques and apparatus describedabove can be used in various combinations.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1. A method of applying a stacked analyte to a channel segmentcomprising: stacking one or more analytes in a channel; detecting avoltage potential in the channel; and, applying an electric field or apressure differential along a channel segment when a selected voltageevent is detected; thereby applying the stacked analytes to the channelsegment.
 2. The method of claim 1, wherein stacking comprises transientstacking or steady state stacking.
 3. The method of claim 1, wherein thechannel comprises a microscale channel.
 4. The method of claim 1,wherein the channel comprises a stacking channel segment or a separationchannel segment.
 5. The method of claim 4, wherein the stacking channelsegment comprises a trailing electrolyte or a leading electrolyte. 6.The method of claim 4, wherein the stacking channel segment comprises atrailing electrolyte and a leading electrolyte, which electrolytescomprise different mobilities.
 7. The method of claim 6, wherein thetrailing electrolyte and the leading electrolyte differ in one or moreof: a pH, a viscosity, a conductivity, size exclusion, an ionicstrength, an ion composition, or a temperature.
 8. The method of claim6, further comprising: adjusting the trailing electrolyte to comprise amobility less than the one or more analytes, or adjusting the leadingelectrolyte to comprise a mobility greater than the one or moreanalytes.
 9. The method of claim 6, further comprising: adjusting thetrailing electrolyte to comprise a mobility greater than one or moresample constituents not of interest, or adjusting the leadingelectrolyte to comprise a mobility less than one or more sampleconstituents not of interest.
 10. The method of claim 1, wherein thechannel comprises a skewing channel segment.
 11. The method of claim 10,wherein the skewing channel segment comprises: a serpentine curve, ahelix, a coil, an angle, or a spiral.
 12. The method of claim 10,wherein the skewing channel segment comprises conditions providing adispersion Peclet number more than 0.1 times a ratio of a skewingchannel length over a skewing channel width.
 13. The method of claim 10,wherein the channel comprises a skewing channel internal width greaterthan a skewing channel depth.
 14. The method of claim 10, wherein theskewing channel segment comprises a travel surface distance on a firstside of the skewing channel greater than a travel surface distance on asecond side of the skewing channel.
 15. The method of claim 10, whereinthe stacking comprises selective isotachophoresis.
 16. The method ofclaim 4, wherein said applying an electric field comprises switchingfrom a substantial lack of current in the separation channel segment anda current in the stacking channel segment to a current in the separationchannel segment and a substantial lack of current in the stackingchannel segment.
 17. The method of claim 16, wherein the substantiallack of current comprises a float voltage or a lack of a completecircuit.
 18. The method of claim 4, wherein the separation channelsegment comprises one or more of: a pH gradient, size selective media,ion exchange media, a hydrophobic media, or a viscosity enhancing media.19. The method of claim 4, further comprising detecting analytes in theseparation channel segment or detecting analytes eluting from theseparation channel segment.
 20. The method of claim 19, wherein saiddetecting analytes comprises monitoring: a conductivity, a fluorescence,a light absorbance, or a refractive index.
 21. The method of claim 1,wherein said stacking comprises consecutively stacking two or moresamples of the analytes in the channel.
 22. The method of claim 21,wherein said stacking the two or more samples comprises: loading a firstsample into a loading channel segment; applying an electric field acrossthe sample, thereby stacking sample analytes; loading a second sampleinto the loading channel segment; and, applying an electric field acrossthe stacked sample analytes and the second sample.
 23. The method ofclaim 22, further comprising flowing the stacked first sample analytestowards the loading channel segment.
 24. The method of claim 1, whereinsaid stacking comprises loading samples of the analytes in a loadingchannel segment comprising a cross-section greater than a stackingchannel segment cross-section.
 25. The method of claim 1, wherein saidstacking comprises loading one or more spacer electrolytes between twoor more sample analytes, which spacer electrolytes comprise a mobilitygreater than a trailing electrolyte and a mobility less than a leadingelectrolyte.
 26. The method of claim 25, wherein one or more of the twoor more sample analytes comprise a stacked sample analyte.
 27. Themethod of claim 25, further comprising adjusting the spacer electrolytesto provide a mobility between mobilities of two or more of the analytes.28. The method of claim 25, further comprising adjusting the mobility ofthe spacer electrolytes by selecting one or more of: a spacerelectrolyte pH, a spacer electrolyte viscosity, or a spacer electrolyteconductivity.
 29. The method of claim 1, further comprising determininga pK of the analytes.
 30. The method of claim 29, further comprisingadjusting the pH of a trailing electrolyte or a leading electrolyte tobe higher or lower than the determined pK.
 31. The method of claim 1,wherein said detecting comprises monitoring a float voltage.
 32. Themethod of claim 1, wherein the voltage event comprises: a voltage peak,a voltage trough, a predesignated voltage, a relative voltage, or a rateof voltage change.
 33. The method of claim 1, wherein said applying anelectric field or pressure differential along the channel segment isautomatic when the voltage event is detected.
 34. The method of claim 1,wherein the analytes comprise one or more of: a protein, a nucleic acid,a carbohydrate, a glycoprotein, a derivitized molecule, or an ion.
 35. Asystem for application of a stacked analyte to a channel segmentcomprising: a channel; an analyte stacking in the channel; and, avoltage detector in electrical contact with the channel, and incommunication with a controller; wherein the controller initiates a flowof electric current in a channel segment or a pressure differentialalong the channel segment when a selected voltage event is detected bythe voltage detector.
 36. The system of claim 35, wherein the analytecomprises one or more of: a protein, a nucleic acid, a carbohydrate, aglycoprotein, a derivatized molecule, or an ion.
 37. The system of claim35, wherein the channel comprises a microscale channel.
 38. The systemof claim 35, wherein the channel comprises a stacking channel segment ora separation channel segment.
 39. The system of claim 38, wherein thestacking channel segment comprises a trailing electrolyte or a leadingelectrolyte, which electrolytes comprise different mobilities.
 40. Thesystem of claim 39, wherein the trailing electrolyte and the leadingelectrolyte differ in one or more of: a pH, a viscosity, a conductivity,a size exclusion, an ionic strength, an ion composition, or atemperature.
 41. The system of claim 39, wherein the trailingelectrolyte comprises a mobility less than a mobility of the analyte ofinterest or a mobility greater than a mobility of a sample constituentnot of interest.
 42. The system of claim 39, wherein the leadingelectrolyte comprises a mobility greater than a mobility of the analyteof interest or a mobility less than a mobility of a sample constituentnot of interest.
 43. The system of claim 38, wherein the separationchannel segment comprises one or more of: a pH gradient, size selectivemedia, ion exchange media, a hydrophobic media, or a viscosity enhancingmedia.
 44. The system of claim 35, wherein the controller comprises alogic device or a system operator.
 45. The system of claim 38, furthercomprising substantial elimination of current in the stacking channelsegment when the voltage event is detected.
 46. The system of claim 38,wherein the channel further comprises a loading channel segment in fluidcontact with the stacking channel segment.
 47. The system of claim 46,wherein the loading channel segment comprises a cross-section greaterthan a stacking channel segment cross-section.
 48. The system of claim46, further comprising a pressure differential across the stackingchannel segment, whereby a stacked sample can be flowed toward theloading channel segment.
 49. The system of claim 46, further comprisinga collector tube through which an analyte sample can flow into theloading channel segment.
 50. The system of claim 38, further comprisinga spacer electrolyte between two or more analyte sample segments in thestacking channel segment.
 51. The system of claim 50, wherein the spacerelectrolyte comprises a mobility between mobilities of two or more ofthe analytes in the sample segments.
 52. The system of claim 35, furthercomprising a float voltage regulator or a switch in electrical contactwith the channel.
 53. The system of claim 35, wherein the voltage eventcomprises one or more of: a voltage peak, a selected voltage, a voltagetrough, a relative voltage, or a rate of voltage change.
 54. The systemof claim 35, wherein the flowing of electric current in the channelsegment or application of the pressure differential along the channelsegment is automatic on detection of the voltage event.
 55. The systemof claim 38, further comprising an analyte detector directed to monitor:analytes in the separation channel segment, or analytes eluted from theseparation channel segment.
 56. The system of claim 55, wherein theanalyte detector comprises: a fluorometer, a spectrophotometer, arefractometer, or a conductivity meter.
 57. The system of claim 35,further comprising a microfluidic chip.
 58. The system of claim 38,wherein the channel comprises a skewing channel segment.
 59. The systemof claim 58, wherein the skewing channel segment comprises: a serpentinecurve, a helix, an angle, or a spiral.
 60. The system of claim 58,further comprising skewing channel segment conditions that provide adispersion Peclet number more than about 0.1 times a ratio of a skewingchannel length over a skewing channel width.
 61. The system of claim 58,wherein the skewing channel segment comprises a skewing channel internalwidth greater than a skewing channel depth.
 62. The system of claim 58,wherein the skewing channel segment comprises a travel surface distanceon a first side of the skewing channel greater than a travel surfacedistance on a second side of the skewing channel.
 63. The system ofclaim 58, wherein the stacking comprises selective isotachophoresis. 64.A method of separating an analyte of interest from a sample constituentnot of interest, the method comprising: stacking the analyte byisotachophoresis in a channel comprising a skewing channel segment; and,flowing the analyte and sample constituent not of interest through theskewing channel segment during or before the isotachophoresis, whereinthe skewing channel segment comprises conditions providing a dispersionPeclet number more than 0.1 times a ratio of a skewing channel lengthover a skewing channel width.
 65. The method of claim 64, wherein theanalytes comprise one or more of: a protein, a nucleic acid, acarbohydrate, a glycoprotein, a derivitized molecule, or an ion.
 66. Themethod of claim 64, wherein the isotachophoresis comprises selectiveisotachophoresis.
 67. The method of claim 64, wherein the skewingchannel comprises: a serpentine curve, a helix, an angle, a coil, or aspiral.
 68. The method of claim 64, wherein the skewing channel segmentcomprises conditions providing a dispersion Peclet number more than theratio of the skewing channel length over the skewing channel width. 69.The method of claim 64, wherein the channel has a greater internal widthat the skewing channel segment.
 70. The method of claim 64, wherein theskewing channel segment comprises a skewing channel internal widthgreater than a skewing channel depth.
 71. The method of claim 64,wherein the skewing channel segment comprises a travel surface distanceon a first side of the skewing channel greater than a travel surfacedistance on a second side of the skewing channel.
 72. The method ofclaim 71, wherein the difference between the travel surface distances ofthe first side and the second side is at least about 25%.
 73. The methodof claim 64, further comprising: detecting a voltage potential in thechannel; and, applying an electric field or a pressure differentialalong a channel segment when a selected voltage event is detected;thereby applying the stacked analytes to the channel segment.
 74. Themethod of claim 73, wherein the channel segment comprises a separationchannel.
 75. A isotachophoresis system comprising: a channel comprisinga skewing channel segment; and, an analyte in the channel stacking byisotachophoresis; wherein the skewing channel segment comprisesconditions that provide a dispersion Peclet number more than 0.1 times aratio of a skewing channel length over a skewing channel width.
 76. Theisotachophoresis system of claim 75, wherein the channel comprises amicro scale channel.
 77. The isotachophoresis system of claim 75,wherein the skewing channel comprises segment: a serpentine curve, ahelix, an angle, or a spiral.
 78. The isotachophoresis system of claim75, wherein the channel has a greater internal width in the skewingchannel segment.
 79. The isotachophoresis system of claim 75, whereinthe skewing channel comprises segment a skewing channel internal widthgreater than a skewing channel depth.
 80. The isotachophoresis system ofclaim 75, wherein the skewing channel segment comprises a travel surfacedistance on a first side of the skewing channel greater than a travelsurface distance on a second side of the skewing channel.
 81. Theisotachophoresis system of claim 75, wherein the dispersion Pecletnumber is more than about the ratio of the skewing channel length overthe skewing channel width.
 82. The isotachophoresis system of claim 75,wherein the analyte comprises one or more of: a protein, a nucleic acid,a carbohydrate, a glycoprotein, a derivitized molecule, or an ion. 83.The isotachophoresis system of claim 75, wherein the isotachophoresiscomprises selective isotachophoresis.
 84. The isotachophoresis system ofclaim 75, wherein the isotachophoresis comprises a leading electrolytecomprising a mobility greater than a mobility of the analyte or atrailing electrolyte comprising a mobility less than the mobility ofanalyte.
 85. The isotachophoresis system of claim 75, wherein theselective isotachophoresis comprises a leading electrolyte comprising amobility less than a mobility of sample constituent not of interest or atrailing electrolyte comprising a mobility greater than the mobility ofthe sample constituent not of interest.
 86. The isotachophoresis systemof claim 75, further comprising: a voltage detector in electricalcontact with the channel, and in communication with a controller;wherein the controller initiates a flow of electric current in a channelsegment or a pressure differential along the channel segment when aselected voltage event is detected by the voltage detector.